This invention relates to a recombinant Sendai virus vector expressing chemokine. More specifically, it relates to a recombinant Sendai virus vector expressing CXC-chemokine. This invention also relates to a method of producing the vector, a pharmaceutical composition comprising it, and a method of inhibiting human immunodeficiency virus (HIV) infection using it.
Sendai virus is a non-segmented negative strand RHA virus in the family Paramyxoviridae. Sendai virus has been known as a biotechnologically useful virus, being widely utilized, especially for the production of heterokaryons and hybrid cells, by taking advantage of viral cell-fusion capacity. Also, Sendai virus-based cell fusing liposomes as a vehicle to deliver foreign genes into cells have being developed. Sendai virus has both infectivity and disseminative capability. In contrast, not only genomic negative strand but also antigenomic positive strand of Sendai viral RNA artificially transcribed in vitro cannot serve as a functional template to form infectious virions when transfected into cells. Recently, a system for efficient reconstitution of viral particles from Sendai viral cDNAs has been established, enabling the gene manipulation of Sendai virus to produce a recombinant Sendai virus (W097/16539, Kato, A. et al., Genes to Cells (1996) 1, 569-579).
Chemokines are basic polypeptides capable of binding to heparin and have leucocyte migration enhancing activity and leucocyte activating activity. Chemokines and chemokine receptors are involved in various biological functions, including human immunodeficiency virus infection, inflammation, and immunoreaction.
There are four subfamilies of chemokines, CXC-, CC-, C-, and CX3C-chemokines, based on the first two cysteine residues. Of the CXC-chemokine subfamily, chemokines having an ELR (Glu-Leu-Arg) motif, such as IL-8, Gro xcex1, Gro xcex2, Gro xcex3, NAP-2, ENA-78, and GCP-2, are potent angiogenic factors, while those with no ELR motif inhibit angiogenesis (Strieter, R. M., J. Biol. Chem. 270, 27348-27357 (1995)). Such chemokines can be used for treat tumors by expressing a chemokine-encoding gene in excess in tumor cells. Furthermore, expression of chemokines in dendritic cells and existence of their receptors have been recently reported (Legler, D. F., J. Exp. Med. 187, 655-660 (1998)). This suggests that cbemokines may be involved in immunological maturation of various T-cells. The CXC chemokines, SDF-1xcex1 and SDF-1xcex2, are known to inhibit replication of T-cell line tropic HIV, and CC chemokines, MIP-1xcex1 and MIP-1xcex2, and RANTES, inhibit macrophage tropic HIV strains.
SDF-1 is suggested to have the most potent T-cell chemotactic activity and the most potent T-cell adhesion inducing activity (Campell et al., Science 279, 381-382 (1998)). It promotes adhesion of thymocytes of the different differentiation stages to tissues and regulates their distribution in the thymus.
These facts suggest that chemokines, particularly SDF-1, can accelerate establishment of immunity modulated by T-cells and can be used to regulate enhancement of immunity against cancer and effects of vaccines.
In general, recombinant highly basic polypeptides or peptides such as chemokines are produced in Escherichia coli with low productivity. Furthermore, E. coli-based production generally requires extensive, multi-step purification of the product before use, and therefore is not always feasible for testing many different, genetically engineered derivatives. Extensive aggregation is often inevitable particularly for such basic polypeptides as chemokines. Thus, chemical synthesis of the original and modified versions has been adopted. This approach is not only laborious, including careful refolding, but also expensive. Purification of recombinant chemokines produced in mammalian and other higher vertebrate cells by recombinant viruses has not been reported.
An objective of this invention is to provide a method of producing recombinant pharmaceutically useful chemokines in large quantity. Another objective of the present invention is to provide a system to produce recombinant chemokines and its medical use.
In order to achieve the above objective, the present inventors made attempts to create recombinant Sendai virus expressing chemokines and found that the recombinant virus produced and accumulated the chemokines expressed in large quantity.
Thus, the present invention provides a recombinant Sendai virus vector expressing chemokine.
The present invention also provides a method of producing chemokine, which comprises inserting at least one chemokine gene into a Sendai virus vector, allowing the vector to produce chemokine, and recovering chemokine.
Furthermore, the invention provides a method of treating human immunodeficiency virus infection, which comprises administering to human subjects a recombinant Sendai virus vector expressing CXC- and CC-chemokines and allowing the vector to express the chemokine in vivo. It also provides a method of treating human immunodeficiency virus infection, which comprises collecting cells from human subjects, infecting the cells with a recombinant Sendai virus vector expressing the chemokine, and giving the infected cells back to the human subjects.